Abstract [en]

The relation between current and voltage, I-V relation, is central to functional analysis of membrane ion channels. A commonly used method, since the introduction of the voltage-clamp technique, to establish the I-V relation depends on the interpolation of current amplitudes recorded at different steady voltages. By a theoretical computational approach as well as by experimental recordings from GABA(A) receptor mediated currents in mammalian central neurons, we here show that this interpolation method may give reversal potentials and conductances that do not reflect the properties of the channels studied under conditions when ion flux may give rise to concentration changes. Therefore, changes in ion concentrations may remain undetected and conclusions on changes in conductance, such as during desensitization, may be mistaken. In contrast, an alternative experimental approach, using rapid voltage ramps, enable I-V relations that much better reflect the properties of the studied ion channels.

Yelhekar, Tushar

Abstract [en]

The overall aim of the present thesis is to clarify the control of intracellular chloride homeostasis in central neurons, because of the critical role of chloride ions (Cl–) for neuronal function. Normal function of the central nervous system (CNS) depends on a delicate balance between neuronal excitation and inhibition. Inhibition is, in the adult brain, most often mediated by the neurotransmitter γ-aminobutyric acid (GABA). GABA may, however, in some cases cause excitation. GABA acts by activating GABA type A receptors (GABAARs), which are ion channels largely permeable to Cl–. The effect of GABAAR-mediated neuronal signaling - inhibitory or excitatory - is therefore mainly determined by the Cl– gradient across the membrane. This gradient varies with neuronal activity and may be altered in pathological conditions. Thus, understanding Cl– regulation is important to comprehend neuronal function. This thesis is an attempt to clarify several unknown aspects of neuronal Cl– regulation. For such clarification, a sufficiently sensitive method for measuring the intracellular Cl– concentration, [Cl–]i, is necessary. In the first study of this thesis, we examined two electrophysiological methods commonly used to estimate [Cl–]i. Both methods, here called the interpolation and the voltage-ramp method, depend on an estimate of the Cl– equilibrium potential from the current-voltage relation of GABA- or glycine-evoked Cl– currents. Both methods also provide an estimate of the membrane Cl– conductance, gCl. With a combination of computational and electrophysiological techniques, we showed that the most common (interpolation) method failed to detect changes in [Cl–]i and gCl during prolonged GABA application, whereas the voltage-ramp method accurately detected such changes. Our analysis also provided an explanation as to why the two methods differ. In a second study, we clarified the role of the extracellular matrix (ECM) for the distribution of Cl– across the cell membrane of neurons from rat brain. It was recently proposed that immobile charges located within the ECM, rather than as previously thought cation-chloride transporter proteins, determine the low [Cl–]i which is critical to GABAAR-mediated inhibition. By using electrophysiological techniques to measure [Cl–]i, we showed that digestion of the ECM decreases the expression and function of the neuron-specific K+ Cl– cotransporter 2 (KCC2), which normally extrudes Cl- from the neuron, thus causing an increase in resting [Cl–]i. As a result of ECM degradation, the action of GABA may be transformed from inhibitory to excitatory. In a third study, we developed a method for quantifying the largely unknown resting Cl– (leak) conductance, gCl, and examined the role of gCl for the neuronal Cl– homeostasis. In isolated preoptic neurons from rat, resting gCl was about 6 % of total resting conductance, to a major part due to spontaneously open GABAARs and played an important role for recovery after a high Cl– load. We also showed that spontaneous, impulse-independent GABA release can significantly enhance recovery when the GABA responses are potentiated by the neurosteroid allopregnanolone. In a final commentary, we formulated the mathematical relation between Cl– conductance, KCC2-mediated Cl– extrusion capacity and steady-state [Cl–]i. In summary, the present thesis (i) clarifies how well common electrophysiological methods describe [Cl–]i and gCl, (ii) provides a novel method for quantifying gCl in cell membranes and (iii) clarifies the roles of the ECM, ion channels and ion transporters in the control of [Cl–]i homeostasis and GABAAR-mediated signaling in central neurons.